Dr. Lo established his independent research program in July of 2008, focusing on melanoma pathogenesis and targeted therapy with support from the UCLA Department of Medicine/Dermatology and the Jonsson Comprehensive Cancer Center. Metastatic cutaneous melanoma is an aggressive malignancy, which lacks effective treatment but harbors a common and druggable target, the V600EB-RAF mutant kinase. Early clinical experience with melanoma patients treated with a novel V600EB-RAF-targeting small molecule, PLX4032, is demonstrating unparalleled tumor response in pre-selected patients with V600EB-RAF-mutant tumors. However, dramatic responses to PLX4032 monotherapy are invariably followed by tumor escape and relapse. The proposed project aims to build a knowledge base for designing combinatorial therapies and tackling drug resistance. We are taking complementary pre-clinical genomic approaches (Aims 1 & 2) in order to dissect the pattern of sensitivity and resistance in melanoma patients treated with PLX4032 (Aim 3). First (Aim 1), we seek to identify melanoma kinases that, together withV600EB-RAF, buffer melanoma against growth arrest/death (i.e., co-dependent growth/survival genes). To construct such a cellular wiring diagram sustaining the survival of V600EB-RAF-positive melanomas, we have established a robust and high-throughput siRNA-based functional screen to identify melanoma kinases whose knockdown sensitizes melanoma cells toV600EB-RAF inhibition. Second (Aim 2), we are modeling PLX4032 resistance in vitro by generating V600EB-RAF-positive melanoma cell lines escaping chronic PLX4032 exposure. Comparing parental PLX4032-sensitive cell lines and their corresponding PLX4032-resistant sub-lines, we propose an integrated genomic approach to discover tumor escape mechanisms to V600EB-RAF targeted inhibition. We believe arming ourselves with knowledge derived from such approaches is critical for a systematic hypothesis-driven study (Aim 3) of precious, paired melanoma samples and cell lines from patients who relapse on PLX4032 in a biopsy-treat-relapse-biopsy protocol. PLX4032 will likely be the first in a series of drugs targeting activated, mutated molecules found specifically only in cancer. Our proposed work will likely provide a critical rational basis to understand the pattern of response/resistance to such therapeutic agents and to design combinatorial therapy to overcome resistance. The proposed comprehensive and objective approaches using melanoma as a model may also offer insights into V600EB-RAF-positive subsets of other malignancies such as colon and ovarian cancers. Furthermore, this grant will provide the applicant with crucial funding to compete for RO1 and other established investigator awards. PUBLIC HEALTH RELEVANCE: Cutaneous melanoma ranks among the fastest rising human malignancies in annual incidence and is highly lethal when detected at advanced stages. Small molecule therapy targeting a common melanoma mutation, V600EB-RAF, is showing unprecedented promise but meets a formidable challenge common to all targeted therapy, cancer resistance and clinical relapse. By understanding the factors determining drug sensitivity and key mechanisms of acquired resistance, we can design better therapies to treat this deadly skin cancer.